JP2005162678A - Lipid for liposome, liposome and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly safe lipid for a liposome, stabilizing the liposome, improving the stability in holding of an including material, and achieving efficient delivery and good targeting thereof; and to provide the liposome obtained by using the lipid, and a method for producing them. <P>SOLUTION: The lipid for the liposome is obtained by dissolving or dispersing a liposome membrane-constituting material containing at least a phospholipid having the transition temperature in carbon dioxide heated to a temperature not lower than the transition temperature of the phospholipid and in the supercritical or subcritical state, adding water to the dissolved or dispersed product, stirring the resultant mixture, reducing the pressure of the mixture to atmospheric pressure, and freeze-drying the aqueous suspension containing the liposome membrane-constituting material. The a water-soluble medicine-containing liposome is obtained by adding the lipid for the liposome to the aqueous solution of the medicine and mixing the resultant product. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a lipid for liposome, a liposome, and a method for producing them, and more specifically, a supercritical material in which a liposome membrane-constituting material containing at least a phospholipid having a phase transition temperature is heated to a temperature higher than the phase transition temperature of the phospholipid. The present invention also relates to a liposome lipid, a liposome, and a production method thereof, which are produced by dissolving or dispersing in carbon dioxide in a subcritical state.

  In order to realize selective and efficient in vivo delivery of specific substances, liposomes composed of lipids similar to biological membranes and considered to be highly safe due to low antigenicity, medicinal substances, contrast-enhancing compounds, etc. A method to encapsulate is being studied. For example, International Publications WO 88/09165, WO 89/00988, WO 90/07491, JP 07-316079 and JP 2003-5596 propose liposomes containing ionic or nonionic contrast agents. In these methods, despite the use of liposomes that are highly safe as materials and have appropriate degradability in vivo, organic solvents such as chloroform, Use a chlorinated solvent such as dichloromethane. Therefore, the above method has not been put into practical use because of the toxic nature of the remaining solvent (see, for example, Patent Document 1).

  On the other hand, lipid-soluble drugs are easily encapsulated in liposomes, but the encapsulated amount depends on other factors and is not necessarily so much. A drug that is a water-soluble electrolyte can be encapsulated in the aqueous phase inside the liposome through the interaction between the charge of the drug and the charge of the charged lipid, but if the drug is a water-soluble non-electrolyte, such means should be used. It cannot be taken. Furthermore, the formed liposome tends to be multi-layered, and the efficiency is poor because the encapsulation rate of medicinal substances is low. As means for efficiently encapsulating such a water-soluble non-electrolyte in the liposome, there are a reverse phase evaporation method and an ether injection method. However, since an organic solvent is used, there still remains a safety problem.

In JP-A-2003-119120 (Patent Document 2), a cosmetic containing a liposome and an external preparation for skin are disclosed.
A method of producing using supercritical carbon dioxide is disclosed, and an example of producing an external preparation for skin in which a hydrophilic medicinal component or a lipophilic medicinal component is encapsulated in liposomes is shown. However, although examples of water-soluble electrolytes have been shown as hydrophilic medicinal ingredients, it was unclear whether water-soluble non-electrolytes could be efficiently encapsulated in liposomes by this method.

Furthermore, there is a problem related to the stability of the liposome itself. Instability that liposomes aggregate or disintegrate during storage, or are phagocytosed by immune system cells before they reach the target site after being administered in vivo, and encapsulated substances leak out of the lipid membrane Instability must also be resolved for practical use.
Japanese Patent Laid-Open No. 7-316079 Japanese Patent Laid-Open No. 2003-119120

  It is an object of the present invention to provide a lipid for liposome for producing a liposome having enhanced retention efficiency as a microcarrier for a medicinal substance, a contrast substance and the like, a liposome using the liposome, and a production method thereof. More specifically, an object of the present invention is to provide a liposome in which a water-soluble drug or the like is encapsulated in a single membrane liposome having a narrow particle size range without using a toxic organic solvent, and a method for producing the liposome.

  The present invention for solving the above problems has the following configuration.

  The method for producing a lipid for liposome of the present invention comprises dissolving a liposomal membrane-constituting substance containing at least a phospholipid having a transition temperature in carbon dioxide in a supercritical or subcritical state heated to the transition temperature of the phospholipid or higher. This is a production method obtained by dispersing, adding water to this, stirring, then reducing the pressure to atmospheric pressure, and lyophilizing an aqueous suspension containing the liposome membrane constituent.

  Prior to the lyophilization, the aqueous suspension is preferably heated to the “transition temperature + 10” ° C. or higher of the phospholipid and then passed through a membrane having pores.

  The lipid for liposome of the present invention is a lipid for liposome obtained by the above production method.

  The method of the present invention is a method for producing a water-soluble drug-containing liposome obtained by adding the above lipid for liposome to an aqueous drug solution and mixing it.

The water-soluble drug-containing liposome of the present invention is a liposome obtained by the above production method.
[Detailed Description of the Invention]
The lipid for liposome of the present invention is obtained by dissolving or dispersing a liposome membrane-constituting material containing at least a phospholipid having a transition temperature in carbon dioxide in a supercritical or subcritical state heated to a temperature higher than the transition temperature of the phospholipid. This is a lipid for liposome obtained by adding water to this and stirring, then reducing the pressure to atmospheric pressure, and lyophilizing an aqueous suspension containing the liposome membrane constituent. The “(phase) transition temperature” of a phospholipid is a temperature that causes a phase transition between both the gel and liquid crystal states that the phospholipid can take. The measurement is by differential thermal analysis using a differential scanning calorimeter (DSC).

Hereinafter, the lipid for liposome of the present invention, the method for producing the lipid for liposome, the method for producing the liposome, the liposome and the use thereof will be described in detail.
Lipid liposomes for liposomes are bilayer vesicles and are generally composed of lipid membranes. The “lipid for liposome” of the present invention and the “liposome membrane constituent material” used in the production method thereof are not particularly limited as long as they are generally used as membrane constituent materials. In general, there are substances that have lipids capable of forming liposomes as essential essential components and coexist as optional components. Optional components include those conventionally used as vesicle-forming components such as sterols as membrane stabilizers, fatty acids or salts thereof as charged substances, and lipid mixed emulsions. Lipids having liposome-forming ability include phospholipids and glycolipids, and phospholipids and derivatives thereof are particularly preferable.

  Preferred neutral phospholipids for liposomes used in the method of the present invention include lecithin, lysolecithin and / or hydrogenated products and hydroxide derivatives obtained from soybean, egg yolk and the like.

Examples of other phospholipids include phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, phosphatidic acid, and more specific examples include dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine ( DSPC)
, Dimyristolphosphatidylcholine (DMPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylserine (DSPS), distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylinositol (DPPI), distearoyl Examples thereof include phosphatidylinositol (DSPI), dipalmitoyl phosphatidic acid (DPPA), distearoyl phosphatidic acid (DSPA), and the like. Of these, phosphatidylcholine is preferred as the main component.

  The lipid for liposome of the present invention is characterized by containing at least a phospholipid having a transition temperature as a liposome membrane constituent. As phospholipids having a phase transition point, dimyristoyl phosphatidylcholine (transition temperature, the same below, 23-24 ° C), dipalmitoylphosphatidylcholine (41.0-41.5 ° C), hydrogenated soybean lecithin (53 ° C), hydrogenated soybean phosphatidylcholine (54 ° C), distearoylphosphatidylcholine (54.1-58.0 ° C), and the like.

  These phospholipids are usually used alone, but may be used in combination of two or more. However, when two or more kinds of charged phospholipids are used, it is desirable to use them between negatively charged phospholipids or between positively charged phospholipids from the viewpoint of preventing liposome aggregation. When neutral phospholipids and charged phospholipids are used in combination, the weight ratio is usually 200: 1 to 3: 1, preferably 100: 1 to 4: 1, and more preferably 40: 1 to 5: 1. Moreover, it is preferable that the alkyl chain length of the fatty acid which comprises a lipid is the same, or the difference in carbon number is 2 or less.

  Examples of glycolipids include glycerolipids such as digalactosyl diglyceride and galactosyl diglyceride sulfate, galactosylceramide, galactosylceramide sulfate, lactosylceramide, sphingoglycolipids such as ganglioside G7, ganglioside G6, and ganglioside G4.

In addition to the above lipids, other substances can be added as necessary as membrane constituents of the liposome. For example, cholesterol, dihydrocholesterol, cholesterol esters, phytosterol, cytocholesterol, stigma cholesterol, campestercholesterol, cholestanol, lanosterol or 2 that act as membrane stabilizers for unstable liposomes that may collapse, aggregate, etc. Sterols such as 1,4-dihydrolanosterol. Further, sterol derivatives such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside have been shown to be effective in stabilizing liposomes (Japanese Patent Laid-Open No. 5-245357).
The amount of sterol used is 0.01 to 0.5 mol, preferably 0.05 to 0.1 mol, per mol of phospholipid. If it is less than 0.01 mol, stabilization by sterol is not exhibited, 0.5
If it is more than the molar amount, the formation of liposomes is inhibited, or even if formed, it becomes unstable.
Cholesterol in the liposome membrane can also serve as an anchor for introducing polyalkylene oxide. Japanese Patent Application Laid-Open No. 09-3093 discloses a novel “functional substance” that can be efficiently immobilized at the end of a polyoxyalkylene chain by a covalent bond and can be used as a liposome-forming component. Cholesterol derivatives are disclosed.

  Other examples of compounds that can be added include dialkyl phosphates such as dicetyl phosphate, which is a negatively charged substance, and aliphatic amines such as stearylamine, as compounds that give a positive charge.

A polymer chain polyalkylene oxide chain (PAO, polyoxyalkylene chain) or polyethylene glycol (PEG) chain, that is, — (CH ₂ CH ₂ O) n-HPEG chain, is attached to the outer surface of the liposome as a component of the liposome membrane. Thus, a new function can be imparted to the liposome. For example, PEGylated liposomes can be expected to have an effect of being hardly recognized by the immune system (for example, reticuloendothelial cells) (in a so-called “stealth” state). Alternatively, it has been clarified that liposomes have a tendency to be hydrophilic, thereby increasing blood stability and maintaining the concentration in blood over a long period of time (Biochim. Biophys. Acta., 1066 , 29-36 (1991)). ).

  Using these properties, organ specificity can be imparted to the liposome. Specifically, since lipid components are likely to be stored in the liver, it is preferable not to use PEG or to use liposomes with a low PEG content when aiming at selective accumulation of the liver. Further, when the particle size is increased to 200 nm or more, the possibility of rapid uptake by the phagocytosis of liver Kupffer cells increases, and it accumulates at the site of the liver. On the other hand, in the case of delivery to other organs, the use of PEGylated liposomes is recommended because introduction of PEG can make the liposomes stealthy and less likely to collect in the liver. Since the hydration layer is formed by the introduction of PEG, the liposome is stabilized and the retention in blood is also improved. By appropriately changing the length of the oxyethylene unit of PEG and the ratio of introduction, the function can be adjusted. As PEG, polyethylene glycol having 10 to 3500 oxyethylene units is preferred. The amount of PEG used is 0.1 to 30% by mass, preferably about 1 to 15% by mass, based on the lipid constituting the liposome.

  A known technique can be used for PEGylation of the liposome. An anchor to which PEG is bound (for example, cholesterol) may be mixed with a phospholipid as a membrane constituent substance to prepare a liposome, and activated PEG may be bound to the anchor. In addition, since the polyethylene glycol group introduced into the liposome surface does not react with the “functional substance”, it is difficult to immobilize the “functional substance” on the liposome surface. Instead, PEG having some modification at the PEG tip may be bound to phospholipid and included in the liposome membrane constituent as a constituent of the liposome membrane.

Instead of the PEG, various known polyalkylene oxide groups,-(AO) n-Y, may be introduced on the outer surface of the liposome. Here, AO represents an oxyalkylene group having 2 to 4 carbon atoms, n is an average added mole number of the oxyalkylene group, and is a positive number of 1 to 2000. Y represents a hydrogen atom, an alkyl group, or a functional functional group.

Examples of the oxyalkylene group having 2 to 4 carbon atoms (represented by AO) include, for example, oxyethylene group, oxypropylene group, oxytrimethylene group, oxytetramethylene group, oxy-1-ethylethylene group, oxy-1,2 -A dimethylethylene group etc. are mentioned. These oxyalkylene groups are groups obtained by addition polymerization of alkylene oxides such as ethylene oxide, propylene oxide, oxetane, 1-butene oxide, 2-butene oxide, and tetrahydrofuran.
n is a positive number of 1 to 2000, preferably 10 to 500, and more preferably 20 to 200.

  When n is 2 or more, the types of oxyalkylene groups may be the same or different. In the latter case, it may be added in a random manner or a block shape. In the case of imparting hydrophilicity to the polyalkylene oxide chain, AO is preferably an ethylene oxide added alone, and in this case, n is preferably 10 or more. When different types of alkylene oxides are added, it is desirable that ethylene oxide is added in an amount of 20 mol% or more, preferably 50 mol% or more. When imparting lipophilicity to the polyalkylene oxide chain, the number of added moles other than ethylene oxide is increased.

Y is a hydrogen atom, an alkyl group or a functional functional group. 1 alkyl as an alkyl group
The aliphatic hydrocarbon group of -5 which may be branched is mentioned. The functional functional group is for attaching a “functional substance” such as sugar, glycoprotein, antibody, lectin, cell adhesion factor to the end of the polyalkylene oxide chain. For example, amino group, oxycarbonylimidazole group, N- A reactive functional group such as a hydroxysuccinimide group may be mentioned.

A phospholipid or compound having a polyalkylene oxide group can be used alone or in combination of two or more. The content is 0.001 to 50 mol%, preferably 0.01 to 30 mol%, more preferably 0.1 to 10 mol%, based on the total amount of the liposome membrane-forming components. If it is less than 0.001 mol%, the expected effect becomes small.

  A known technique can be used to introduce the polyalkylene oxide chain into the liposome. For example, an anchor (for example, cholesterol) to which polyalkylene oxide is bonded is mixed with a phospholipid as a liposome membrane constituting substance to prepare a liposome, and activated polyalkylene oxide is bonded to the anchor. In such a method, it is necessary to perform a multi-step chemical reaction on the surface of the liposome membrane after preparing the liposome, the amount of introduction of the target “functional substance” is limited to a low level, and byproducts and impurities due to the reaction are reduced. There are problems such as contamination and large damage to the liposome membrane.

As a preferable alternative to this, it is preferable to prepare liposomes in advance by including a phospholipid polyalkylene oxide (PEO) derivative in the raw material phospholipids. For this purpose, modified phospholipids such as polyethylene oxide (PEO) derivatives such as phosphatidylethanolamine, such as distearoyl phosphatidylethanolamine polyethylene oxide (DSPE-PEO) have been proposed (Japanese Patent Laid-Open No. 7-165770). . Furthermore, JP 2002-37883 A discloses high-purity polyalkylene oxide-modified phospholipids for producing water-soluble polymer-modified liposomes with increased blood retention. It is described that when a polyalkylene oxide-modified phospholipid having a low monoacyl body content is used in preparing a liposome, the temporal stability of the liposome dispersion is good. In the prior art, liposomes are almost always composed of lipid components such as phospholipids, sterols, and lecithins, together with solvents such as chloroform, dichloromethane, ethyl ether, carbon tetrachloride, ethyl acetate, dioxane, and THF. It is prepared by mixing, dispersing and dissolving in a container to obtain a mixed lipid. Such preparations of liposomes always contain an organic solvent. In order to remove the remaining organic solvent, the present situation requires a multi-step process and a long-time treatment. In addition, in order to produce liposomes with excellent storage stability and uniformity on an industrial scale, the (mixed) lipids for liposomes, which are intermediate raw materials for liposomes, are homogenized into properties that are easy to use for industrial production, and water dispersibility is improved. It needs to be processed into a good shape. As a method for this, JP-A-9-87168 discloses that a liposome membrane constituent substance is dissolved in a homogeneous solvent containing a water-insoluble volatile organic solvent, and water or an aqueous solution is added thereto, followed by stirring. A method has been proposed in which the organic solvent is distilled off and the remaining liposome membrane constituent mixed aqueous phase is freeze-dried. Even in such a method, the step of distilling off most of the organic solvent is essential, and it is inevitable that the operation becomes complicated. The remaining organic solvent is removed by freeze-drying, but it is not easy to completely remove the chlorinated organic solvent used. Chlorine organic solvents are most worried about adverse effects on living organisms, specifically side effects.

The production method of the present invention for preparing liposome lipids does not substantially use an organic solvent when dispersing or dissolving liposome membrane constituents such as phospholipids. Instead, supercritical carbon dioxide or subcritical carbon dioxide is used as a solvent for dispersing and dissolving the liposome membrane constituent material. Carbon dioxide has a critical temperature of 31.1 ° C and a critical pressure of 73.8 bar.
It is suitable for the reason that it is relatively easy to handle, is innocuous to the human body even if it remains because of an inert gas, and a high-purity fluid can be easily obtained at low cost. Furthermore, the liposome produced by this method has various favorable characteristics and advantages for encapsulating a water-soluble drug including a medicinal substance and an iodine compound as an X-ray contrast agent, as will be described later.

When preparing lipids for liposomes using supercritical or subcritical carbon dioxide, liposome membrane constituents, mainly phospholipids, are mixed with carbon dioxide in a supercritical state (including subcritical state) and dispersed. Or it becomes necessary to dissolve. Such supercritical carbon dioxide is excellent in solubility because it easily penetrates into the substance. At that time, it is desirable to use one or more alcohols such as lower alcohols, glycols, and glycol ethers as a solubilizer (cosolvent) in order to further improve the solubility of the lipid membrane component. For example, alcohols may be used as dissolution aids (cosolvents) in a proportion of 0.1 to 10% by mass, preferably 1 to 8% by mass of supercritical carbon dioxide. Among these, a more preferred solubilizer is a lower alcohol, particularly ethanol, from the viewpoint of safety and ease of removal.

The temperature of carbon dioxide in the supercritical state (including subcritical state) used in the production method of the present invention is usually 25 to 100 ° C, preferably 31 to 80 ° C. A particularly suitable temperature within such a temperature range is at or above the phase transition temperature of the phospholipid used. The pressure is usually 50 to 500 atmospheres,
Preferably it is the range of 100-400 atmospheres. Specifically, when the temperature is determined, the pressure is used to establish a supercritical state (including a subcritical state) of carbon dioxide. In addition, because it is not a method of forcibly aggregating lipids in water by applying high heat, lipids are denatured by high heat, for example, phospholipids are hydrolyzed to give lyso forms, and unsaturated components are peroxidized. There is no such problem.

The preparation of lipid for liposomes based on the method of the present invention is specifically performed as follows. Liquid nitrogen dioxide is added in the presence of the liposome membrane constituent material, and as described above, the temperature is higher than the transition temperature of the phospholipid having the transition temperature, and the carbon dioxide is in the supercritical or subcritical state under suitable pressure. To do. As a liposome membrane constituent, in addition to phospholipids having a transition temperature as a main component, other phospholipids, preferably polyalkylene oxide-modified phospholipids, compounds having polyalkylene oxide groups, compounds having polyethylene glycol groups, sterols Are added together with at least one compound selected from above and dispersed or dissolved under stirring. In that case, the said dissolution aid can be used together as needed. Subsequently, water is added to the resulting lipid mixture to form an aqueous phase / carbon dioxide emulsion. Further stirring is continued and water is added continuously. It is considered that the phase transition of the system occurs with the increase of the aqueous phase, and excess carbon dioxide is separated from the carbon dioxide / water emulsion through a two-phase system of water / carbon dioxide emulsion + carbon dioxide / water emulsion. Since the liposome membrane constituents are presumed to have phase-inverted to the aqueous phase, when the system is finally depressurized to atmospheric pressure and carbon dioxide is discharged, the aqueous suspension of liposome lipids encapsulates water. (Lipid-dispersed aqueous phase) is formed.

  The produced aqueous suspension may be frozen as it is using liquid nitrogen or the like and freeze-dried, or may be freeze-dried after being separated from the aqueous phase by centrifugation. The lipid for liposome of the solid powder thus obtained is a mixture of phospholipid and coexisting substance, but is suitably used as an intermediate raw material for liposome production. In addition, when it preserve | saves in the state of mixed lipid, without making solid powder as mentioned above, since phase separation etc. arise, storage stability is also not good. Further, before mixing with the drug solution to be encapsulated in the liposome, the water-soluble drug to be mixed is diluted because water is already contained in the mixed lipid for liposome. Therefore, it is disadvantageous when it is desired to encapsulate the drug at a high concentration.

As a preferred embodiment of the present invention, it is desirable that an aqueous suspension of lipids for liposomes is heated to “transition temperature + 10” ° C. or higher before lyophilization to permeate the membrane with pores. Specifically 0.
A filtration membrane having a pore size of 1 to 0.4 µ is passed. This “transition temperature + 10” ° C. or higher is generally from 35 ° C. to 65 ° C., depending on the type and composition of the phospholipid used. In the operation of pressure extrusion filtration, when heated in this manner, the phospholipid having a transition temperature is in a liquid crystal state, and the fluidity is increased. Therefore, filter sterilization can be performed at a relatively low pressure without causing clogging of the filter. For the extrusion filtration of the dispersion liquid, the filter is forcibly permeated through various hydrostatic extruders, such as “Extruder” (trade name, manufactured by NOF Liposome), “Liponizer” (trade name, manufactured by Nomura Microscience), etc. Let As the filter, a polycarbonate type, a cellulose type or the like can be appropriately used. As a result, a lipid for liposomes having a uniform orientation of lipid molecules and good water dispersibility can be obtained.

The method of lyophilization is not particularly limited. For example, methods such as an indirect heating freezing method, a refrigerant direct expansion method, a heat medium circulation method, a triple heat exchange method, and an overlapping refrigeration method can be mentioned. As a freeze-drying apparatus, any commercially available freeze-dryer can be used. Furthermore, lyophilization conditions can be appropriately selected by those skilled in the art and are not particularly limited. For example, the lyophilization temperature is about -190 to -4 ° C, preferably -120 to -20 ° C, more preferably about -80 to -60 ° C. The pressure is about 0.1 to 35 Pa, preferably 1 to 15 Pa, and more preferably about 5 to 10 Pa. The lyophilization time is, for example, 2 to 48 hours, preferably 6 to 36 hours, and more preferably 16 to 26 hours.
Preparation of liposomes encapsulating a water-soluble drug A method for preparing liposomes used in a drug delivery system or the like in the production method of the present invention is described in detail below.

Various methods have been proposed so far for preparing liposomes. When the production method is different, the shape and properties of the final liposome are also often significantly different (Japanese Patent Laid-Open No. 6-80560). Therefore, the production method is appropriately selected according to the desired form and characteristics of the liposome.

In the production method of the present invention, the liposome lipid obtained by the above method is added to an aqueous solution containing a water-soluble drug and mixed to form a liposome suspension encapsulating the water-soluble drug. If necessary, a formulation aid may be added to the aqueous solution along with the drug to be encapsulated. 1 g of liposome membrane constituent substance is added to 1 to 1000 ml of an aqueous solution (drug aqueous solution) containing a water-soluble drug and suspended. By applying a physical force to the suspension using an emulsifier, a high-speed stirrer or the like to obtain a dispersion, liposomes encapsulating a water-soluble drug are formed.

  Examples of the aqueous medium for preparing the aqueous drug solution include water such as distilled water, pharmacopoeia water and pure water, as well as aqueous solutions containing physiological saline, various buffers, salts and the like. The above water-soluble drugs are drugs that are encapsulated in the aqueous phase in the liposome membrane, and include medicinal substances, contrast substances, physiologically active substances such as antibodies, enzymes, hormones, and various formulation aids. Of these, medicinal substances or contrast substances and various formulation aids are desirable. The medicinal substance includes various pharmaceutical substances, and is preferably a water-soluble non-electrolytic substance. Specific examples include anticancer agents, antibiotics, anti-inflammatory agents, thrombolytic agents, antianginal agents, blood circulation promoters and the like.

  The “formulation aid” is added together with a medicinal substance at the time of formulation, and various substances are appropriately used based on conventional drug production techniques. Specifically, various physiologically acceptable buffers, chelating agents, and if necessary, osmotic pressure regulators, stabilizers, viscosity regulators, antioxidants such as α-tocopherol, paraoxybenzoic acid And preservatives such as methyl.

The various buffering agents include water-soluble amine buffering agents, phosphate buffering agents, citrate buffering agents, carbonate buffering agents, and acetic acid buffering agents. Among these, a water-soluble amine buffer is preferable. Specific examples include tris, ethanolamine, diethanolamine, triethylamine, morpholine, and the like. In particular, Tris is preferable for the reason described later. Examples of the chelating agent include EDTA, EDTANa ₂ -Ca (disodium calcium edetate), EDTANa ₂ , hexametaphosphoric acid, and the like that are approved for pharmaceutical use. In the end, water-soluble amine buffers and chelating agents are particularly preferably used.

  The above-mentioned liposome preparation method using supercritical or subcritical carbon dioxide has a higher rate of uniform liposome production, encapsulation rate of the encapsulated substance, and residual rate of encapsulated substance in the liposome than the conventional method. (Patent Document 2). Furthermore, since single-membrane liposomes can be prepared in a single step in a short time, application on an industrial scale is also possible. Therefore, the present method that can efficiently encapsulate a water-soluble drug in a liposome without substantially using an organic solvent is a useful method for producing a liposome preparation having the above-mentioned properties.

In order to align the particle size distribution of the liposomes to be produced in a narrower range, the liposome suspension may be forcibly permeated through a filtration membrane having a fixed pore size, preferably a polycarbonate membrane or a cellulose membrane. In this case, uniform liposomes having an optimum size of 100 to 300 nm as the center particle size of a single membrane are efficiently prepared by passing through a hydrostatic extrusion apparatus equipped with a filter having a pore size of 0.1 to 0.4 μm as a filtration membrane. be able to. Specifically, various hydrostatic extrusion apparatuses such as “Extruder” (trade name, manufactured by NOF Liposome), “Riponizer” (trade name, manufactured by Nomura Microscience) and the like are used. Since the lipid for liposome of the present invention has a uniform lipid membrane structure with regular orientation, the particle size of the liposome without causing clogging of the filter even in a highly viscous liposome suspension encapsulating a water-soluble drug. It is possible to prepare liposomes with uniform alignment. The extrusion filtration method is described in, for example, Biochim. Biophys. Acta 557, 9 (1979). Incorporation of such an “extrusion” operation step has the advantage that, in addition to the above sizing, it is possible to exchange liposome dispersions, remove undesirable substances, and filter sterilization. Subsequently, the liposome may be purified by removing unretained drug by a method such as centrifugation, ultrafiltration, or gel filtration. Operations such as concentration and dilution may optionally be performed.

In the water-soluble drug-containing liposome produced by the method of the present invention, as described later, a water-soluble drug such as a medicinal substance or a contrast substance is added at an appropriate weight ratio to the liposome membrane lipid weight from the viewpoint of retention stability. Enclosed inside. Furthermore, the retention stability of the water-soluble drug in the liposome is also achieved by the fact that the liposome has a central particle size of usually 50 to 300 nm and is substantially a single membrane. Single-membrane liposomes are liposomes substantially composed of a unilamellar vesicle in which a phospholipid bilayer is formed as one layer. Here, “substantially” means that the liposome is composed of a phospholipid bilayer in which the replica is generally recognized as one layer in the following transmission electron microscope (TEM) observation by the Freeze fracture replica method. It means that That is, for the observed traces of particles left on the carbon film, the one without a step was determined as a single film, and the one with two or more steps was determined as a “multilayer film”. According to the production method of the present invention, a monolayer liposome containing at least 80%, preferably 90% or more of all liposomes contained in a medicinal substance or contrast medium can be obtained.

Such a single membrane liposome can be efficiently produced by a phase separation method using water using the supercritical carbon dioxide as a solvent for lipids. It has been shown that the liposome preparation method using supercritical carbon dioxide can prepare large unilamellar liposomes (particle size, 0.1 to 1.2 μm) with high retention efficiency of water-soluble substances (Pharm. Tech. Japan 19 ( 5) 819-828 (2003)). On the other hand, in the conventional method for preparing liposomes, liposomes composed of multilamellar vesicles (MLV) often exist in a considerable proportion. Therefore, in order to increase the ratio of single membrane liposomes, further operations such as irradiation with ultrasonic waves or passing through a filter having a fixed pore size many times are required. Single membrane liposomes also have the advantage that the dose of liposomes, in other words, the amount of lipids administered does not increase compared to MLV.

Single membrane liposomes, particularly large unilamellar veislcles (LUV), have the advantage of providing a large encapsulation capacity compared to multilamellar liposomes. The water-soluble drug-containing liposome produced by the method of the present invention has a particle size of 200-1000 nm.
It is located between UV and SUV (Small unilamellar vesicles), which are small unilamellar liposomes having a particle size of less than 50 nm. For this reason, the retention volume is also larger than that of SUV, and the trapping efficiency of the water-soluble non-electrolyte medicinal substance or contrast substance, in other words, the encapsulation efficiency, is remarkably excellent as described later. In addition, unlike MLV and LUV, they are not taken up by reticuloendothelial cells and rapidly disappear from the bloodstream.

  The size of liposome particles and their distribution are closely related to the high blood retention, targeting, and delivery efficiency aimed at by water-soluble drug-containing liposomes produced by the method of the present invention. The particle size can be measured by freezing and breaking replica method. Here, the “center particle size” refers to the particle size having the highest appearance frequency in the particle distribution. The adjustment of the particle size can be carried out according to the formulation or process conditions. For example, when the above supercritical pressure is increased, the liposome particle size formed is reduced.

  As described above, sizing of the particle size is important for imparting passive targeting ability to the liposome. Japanese Patent No. 2619037 describes that by excluding liposomes having a particle size of 3000 nm or more, inadequate retention in lung capillaries is avoided.

The center particle size of the water-soluble drug-containing liposome produced by the method of the present invention is usually 50 to 300 nm, preferably 50 to 200 nm, more preferably 50 to 130 nm. The particle size can be appropriately set according to the purpose of treatment, diagnosis, and X-ray imaging. For example, 110-130 nm is particularly preferred for selective delivery to the tumor site. It is possible to concentrate the drug substance or contrast agent selectively on the cancer tissue by uniformly arranging the liposome particle size within the range of 100 to 200 nm, more preferably 110 to 130 nm. This is known as the “EPR effect”. The pores of the neovascular wall in solid cancer tissue are abnormally larger than the pore size of the capillary wall window (fenestra) of normal tissue, less than 30-80 nm, even with molecules of about 100 nm to about 200 nm in size Leaks from. That is, the EPR effect is due to the fact that the neovascular wall in cancer tissue is more permeable than the microvascular wall in normal tissue.
Liposomes and uses thereof The water-soluble drug-containing liposomes produced by the method of the present invention are substantially monolayer liposomes with a narrow particle size range, and are particularly improved in stability in physiological saline. Yes. The liposome of the present invention can be used for various uses such as a pharmaceutical carrier, a test agent, a diagnostic agent, a sensor, and an immobilized catalyst. A suitable application for exerting the characteristics is the delivery of a medicinal substance to a target site utilizing the stability and retention in blood. A preparation for use in the above-described application and including the water-soluble drug-containing liposome of the present invention is referred to as “liposome preparation” in the present specification. Liposome preparations include formulation aids or additives that have been used as preparation techniques, such as stabilizers, preservatives, antioxidants, osmotic pressure adjusting agents, and the like. Also in its form is an aqueous medium (a solvent that dissolves medicinal substances, formulation aids, etc. based on water, water such as distilled water, pharmacopoeia water, pure water, physiological saline, various buffers, salts Etc.) and a liquid preparation dispersed or suspended.

When a water-soluble non-electrolytic substance, preferably a medicinal substance or a contrast substance is encapsulated in a microcarrier called a liposome, the dose of lipid must be considered in addition to the delivery efficiency and retention stability of the delivery substance. As the amount of lipid increases, the viscosity of the liposome preparation increases. Regarding the amount of the medicinal substance or contrast substance encapsulated in the liposome, the medicinal substance or contrast substance in the aqueous solution encapsulated in the liposome is 1 to 10, preferably 3 to 8, relative to the liposome membrane lipid weight. Preferably, it is contained in a weight ratio of 5 to 8.

When the weight ratio of the medicinal substance or contrast substance encapsulated in the aqueous phase in the liposome is less than 1, it is necessary to inject a relatively large amount of lipid, resulting in poor delivery efficiency of the medicinal substance. . According to the description of Japanese Patent No. 2619037, even if the ratio is 1, it was a high value from the technical level at that time. Since the viscosity of the liposome preparation solution depends on the lipid amount of the liposome, the superiority of the single membrane liposome with excellent retention volume and encapsulation efficiency is clear. Conversely, if the weight ratio of the medicinal substance or contrast material to the liposome membrane lipid weight exceeds 10, the liposome will also be structurally unstable, and the diffusion or leakage of the medicinal substance or contrast material outside the liposome membrane will be in storage. Or even after being injected into a living body. In addition, even if 100% encapsulation is achieved immediately after the liposome suspension drug is produced and isolated, the encapsulated components decrease as soon as possible based on destabilization due to the osmotic pressure effect. (Japanese National Publication No. 9-505821).

The viscosity of the liposome preparation liquid is 6 cPa or less, preferably 0.9 to 3 cPa at 37 ° C.
Furthermore, it is encapsulated in the liposome in the form of an isotonic solution or suspension with respect to the osmotic pressure in the body so that the liposome is stably maintained in the body after administration. The preferred pH range of the liposome preparation is 6.5 to 8.5, more preferably 6.8 to 7.8 at room temperature. When the liposome-encapsulating substance is a water-soluble medicinal substance or a contrast substance having a multi-hydroxyl group, a preferable buffer is a buffer having a negative temperature coefficient as described in US Pat. No. 4,278,654. The amine-based buffer has properties that satisfy such requirements, and tris (trometamol) is particularly preferable. This type of buffer has a low pH at the autoclave temperature, which increases the stability of the liposomal formulation in the autoclave, while returning to a physiologically acceptable pH at room temperature. In this case, there is no restriction of miniaturizing the liposome particle size as in filtration sterilization. Therefore, in order to produce a sterile preparation for injection, it is very convenient to be able to autoclave the liposome preparation, and storage stability and the like can be ensured. However, filtration sterilization should be performed on liposomes to which autoclave sterilization cannot be applied.

  To obtain an isotonic solution or suspension as a liposome formulation, liposomes are dissolved or suspended in a medium at a concentration that provides an isotonic solution. If the formulation alone cannot provide an isotonic solution, other non-toxic water-soluble substances such as salts such as sodium chloride, mannitol, glucose, sucrose, sorbitol so that an isotonic solution or suspension is formed. Sugars such as may be added to the aqueous medium.

An example of a more preferable embodiment of a liposome preparation having improved drug delivery property and retention efficiency of the liposome encapsulating substance has a polyalkylene oxide chain and / or sterol in the lipid membrane, and a central particle size of 50 to 200 nm. And the liposome contains a water-soluble drug in a weight ratio of 3 to 8 with respect to the weight of the lipid membrane. The liposome produced by the method of the present invention aims to stabilize the structure of the liposome and maintain the retention of the encapsulated substance, and enhances the blood retention of the liposome. This can achieve efficient delivery of medicinal substances or contrast substances as well as good targeting.

The method for producing lipids for liposomes according to the present invention employs a method of preparing liposomes by dissolving phospholipids or the like in supercritical carbon dioxide, so that it is not necessary to use a toxic solvent, particularly a highly toxic chlorinated solvent. .
The liposome lipid of the present invention has a uniform orientation of liposome membrane constituents such as phospholipids and good water dispersibility.

Since the liposome according to the method of the present invention is a substantially monolayer liposome having a uniform center particle size, the encapsulation rate of the water-soluble drug as the encapsulated substance is improved. Furthermore, stabilization of the liposome structure and retention stability of the encapsulated substance are improved.

〔Example〕
Hereinafter, the present invention will be described more specifically based on examples. The present invention is not limited in any way by such examples.

Dipalmitoylphosphatidylcholine (DPPC) 40mg, Adeka's "Pluronic F-68" 1.2mg, oleic acid 0.4mg, stainless steel pressure vessel (with internal volume) with a sapphire glass viewing hole 50 ml) with a stir bar and covered. The pressure vessel was placed on a magnetic stirrer, and the inside of the pressure vessel was heated to 60 ° C. while rotating the stirring bar of the pressure vessel. Subsequently, 13 g of liquid carbon dioxide at a pressure of 5 MPa was added. By slowly reducing the internal volume with a piston, liquefied carbon dioxide was made into a supercritical fluid, and was further pressurized slowly to a pressure of 20 MPa. Thereafter, the inside of the pressure vessel was stirred with a stir bar for a while, and then 5 ml of water for pharmacopoeia injection was poured into the pressure vessel over about 50 minutes using a liquid chromatography pump. Thereafter, carbon dioxide in the system was gradually discharged and returned to normal pressure to obtain a liposome dispersion. This operation was repeated 4 times.

The first liposome was heated again to 60 ° C., filtered through a mixed cellulose filter manufactured by Advantech having a pore size of 1 μm, and then filtered six times through a mixed cellulose filter manufactured by Advantech having a pore size of 0.45 μm. This was lyophilized. Thereafter, 5 ml of X-ray contrast agent “Oipamidol 150” manufactured by Konica Minolta MG Co., Ltd. was added and stirred several times to obtain Noliposome dispersion liquid 1 for X-ray contrast agent.

The second liposome was heated again to 60 ° C., filtered through a mixed cellulose filter manufactured by Advantech with a pore size of 1 μm, and then filtered three times with a mixed cellulose filter manufactured by Advantech with a pore size of 0.45 μm. Filtration was performed 6 times with a mixed cellulose filter manufactured. This was lyophilized. Thereafter, 5 ml of X-ray contrast medium “Oipamidol 150” manufactured by Konica Minolta MG Co., Ltd. was added and stirred several times to obtain Noliposome Dispersion Liquid 2 for X-ray contrast medium.

The third liposome was heated again to 60 ° C., filtered through an Advantech mixed cellulose filter with a pore size of 1 μm, then mixed three times with an Advantech mixed cellulose filter with a pore size of 0.45 μm, and mixed with an Advantech company with a pore size of 0.3 μm. Filtration was performed 3 times with a cellulose filter and 6 times with a mixed cellulose filter manufactured by Advantech having a pore size of 0.2 μm.
This was lyophilized. Thereafter, 5 ml of an X-ray contrast agent “Oipamidol 150” manufactured by Konica Minolta MG Co., Ltd. was added and stirred several times to obtain Noliposome dispersion 3 for X-ray contrast agent.

The fourth liposome was heated again to 60 ° C., filtered through an Advantech mixed cellulose filter with a pore size of 1 μm, and then three times with an Advantech mixed cellulose filter with a pore size of 0.45 μm, manufactured by Advantech with a pore size of 0.3 μm. 3 with mixed cellulose filter
The filtration was performed 3 times with a 0.2 μm Advantech mixed cellulose filter and 6 times with a 0.1 μm Advantech mixed cellulose filter. This was lyophilized. Thereafter, 5 ml of X-ray contrast medium “Oipamidol 150” manufactured by Konica Minolta MG Co., Ltd. was added and stirred several times to obtain a liposome dispersion 4 for X-ray contrast medium.

0.5 g of each of the above-mentioned liposome dispersions 1 to 4 was placed in a dialysis membrane, dialyzed 3 times in physiological saline, then placed in ethanol / water 3/1, and the liposomes were broken and encapsulated. When the contrast agent concentration was measured with a spectrophotometer, it was found that 9 to 15% of the drug was included.

Hydrogenated soybean phosphatidylcholine 49mg, N- (carbonyl-methoxypolyethyleneglycol 2000) -1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt 15.95mg, cholesterol 15.95mg, peephole in saffia glass And placed in a stainless steel pressure vessel (internal volume 50 ml) with a movable piston together with a stirrer and capped. The pressure vessel was placed on a magnetic stirrer, and the inside of the pressure vessel was heated to 75 ° C. while rotating the stirring bar of the pressure vessel. Subsequently, 13 g of liquid carbon dioxide at a pressure of 5 MPa was added. By slowly reducing the internal volume with a piston, liquefied carbon dioxide was made into a supercritical fluid, and was further pressurized slowly to a pressure of 20 MPa. Thereafter, the inside of the pressure vessel was stirred with a stir bar for a while, and then 5 ml of water for pharmacopoeia injection was poured into the pressure vessel over about 50 minutes using a liquid chromatography pump. Thereafter, carbon dioxide in the system was gradually discharged and returned to normal pressure to obtain a liposome dispersion.

The liposome dispersion was heated again to 60 ° C., filtered through a mixed cellulose filter manufactured by Advantech with a pore size of 1 μm, and then three times through an advantech mixed cellulose filter with a pore size of 0.45 μm, manufactured by Advantech. 3 with mixed cellulose filter
The filtration was performed 3 times with a 0.2 μm Advantech mixed cellulose filter and 6 times with a 0.1 μm Advantech mixed cellulose filter. This was lyophilized. Thereafter, 5 ml of X-ray contrast medium “Oipamidol 150” manufactured by Konica Minolta MG Co., Ltd. was added and stirred several times to obtain a liposome dispersion for X-ray contrast medium.

  Place 0.5 g of the above liposome dispersion in a dialysis membrane, dialyze three times in physiological saline, and then put in ethanol / water, 3/1 to break the liposomes and spectroscopically analyze the concentration of the contrast medium contained. When measured with a photometer, it was found that 9-15% of the drug was encapsulated.

Claims (5)

  A liposome membrane-constituting material containing at least a phospholipid having a transition temperature is dissolved or dispersed in carbon dioxide in a supercritical or subcritical state heated to a temperature higher than the transition temperature of the phospholipid, added with water, and then stirred. And then reducing the pressure to atmospheric pressure and freeze-drying the aqueous suspension containing the liposome membrane-constituting substance, thereby producing a lipid for liposomes.   2. The aqueous suspension is heated to a “transition temperature + 10” ° C. or higher of the phospholipid before the freeze-drying, and then passed through a membrane having pores. A method for producing lipids for liposomes.   Lipids for liposomes obtained by the production method according to claim 1 or 2.   A method for producing a water-soluble drug-containing liposome obtained by adding the lipid for liposome according to claim 3 to an aqueous drug solution and mixing.   A water-soluble drug-containing liposome obtained by the production method according to claim 4.